User:Douglas M. Fox/Notebook/AU CHEM-571 F2011 Lab Support/2014/09/16: Difference between revisions
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==Tasklist== | ==Tasklist== | ||
* If your AuNP colloid solution has precipitated, remake a 30:1 Au:protein (or lower Au) solution | * If your AuNP colloid solution has precipitated, remake a 30:1 Au:protein (or lower Au) solution | ||
* Prepare | * Prepare 500 mL 50 mM NaCl | ||
* Prepare 1 L 50 mM Glycine buffer, pH 3.5 | * Prepare 1 L 50 mM Glycine buffer, pH 3.5 | ||
**As shown below, I used 0.4 mL of 1 M HCl to make 100 mL of buffer | |||
* Prepare 25 mL 1 g/L Lysozyme | * Prepare 25 mL 1 g/L Lysozyme | ||
* Dialyze about 4 mL of lysozyme in about 3 in of dialysis tube | * Dialyze about 4 mL of lysozyme in about 3 in of dialysis tube | ||
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**Prepare 1 25k g/mol MWCO tubing for Glycine soak | **Prepare 1 25k g/mol MWCO tubing for Glycine soak | ||
*Dialyze 4 mL of your colloid solution in 3500 g/mol MWCO tubing for Glycine soak | *Dialyze 4 mL of your colloid solution in 3500 g/mol MWCO tubing for Glycine soak | ||
*Construct pH and conductivity calibration curve for both NaCl and Glycine buffer | |||
**For each solution, prepare 2x, 5x, 10x, 20x, 50x, 100x, 200x, and 500x dilutions | |||
**'''Why don't we need anything lower than 100 μM?''' | |||
**You will need 12 - 15 mL of each solution. Consider serial dilutions for each order of magnitude. | |||
*Prepare 12 - 15 mL of 0.25 μM HCl from your HCl stock solution | |||
*Measure the pH and conductivity of this dilution | |||
*Use multi-well dialysis apparatus to dialyze lysozyme against: | |||
**50 μM, 25 μM, 2.5 μM, and 0.25 μM glycine buffer | |||
**fill fifth well with 0.25 μM HCl | |||
**before loading wells, you will wet a dialysis sheet and clamp it between the plastic plates | |||
**fill one side with 1 mL of lysozyme and the other side with 1 mL of your buffer | |||
*Analyze inside and outside dialysis membranes (pH, conductivity, Bradford) tomorrow while you wait for electrophoresis | |||
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< | ==Dialysis Protocol== | ||
#Cut about a 3" length of dialysis tubing | |||
#*25,000 MWCO tubing is stored in an azide solution to prevent mold (use care; NaN<sub>3</sub> is toxic) | |||
#*keep the 25,000 MWCO tubing wet to prevent pore shrinkage | |||
#wash cut tubing with DI water, both inside and out | |||
#*3,500 MWCO and 15,000 MWCO are dry and need to be wet prior to filling | |||
#*25,000 MWCO needs to be rinsed to remove the sodium azide | |||
#flatten tubing and remove as much residual water as possible with gloves fingers | |||
#clamp one end 1/4 - 1/2" from edge | |||
#open other end and transfer your '''measured''' protein solution inside | |||
#carefully flatten open end and clamp, making sure no liquid escapes | |||
#rinse with DI water, particularly the ends, which may have residual protein solution on it | |||
#place into a 150 mL beaker (or 250 mL or 400 mL) | |||
#measure out 100 - 200 mL of your dialyzing solution | |||
#label beaker, cover with parafilm, and leave in your drawer overnight | |||
#*we may use the shaker on low speed to help prevent gradients during dialysis | |||
#*you should have 8 solutions when you are finished | |||
Revision as of 19:02, 15 September 2014
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Tasklist
Dialysis Protocol
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